ABSTRACT Trauma or congenital defects of bone, including the craniofacial skeleton, are particularly damaging to physical and psychological well-being. There is an unmet clinical need to improve existing technologies designed to enhance bone formation: to treat traumatic bone injury, reverse age-induced bone loss, and relieve symptoms of heritable disorders of bone metabolism. Notch is a developmental cell-cell signaling pathway with myriad functions that is highly conserved through evolution. Coordinated changes in Notch ligand/receptor presentation alter bone metabolism in a manner dependent on cellular origin and differentiation state. We hypothesize that contextual interactions between the 5 different Notch ligands and the 4 different Notch receptors regulate cell fate decisions driving bone healing, and that Notch ligand gain-of-function may be used to promote therapeutic intramembranous bone formation. This study aims to answer significant yet unexplored questions concerning the role of Notch signaling in adult stem cell biology: 1) What are the specific Notch ligand/receptor interactions driving osteoblastogenesis? 2) How are Notch ligand deficiencies compensated, and what is the resulting bone phenotype? 3) Can modulation of Notch signaling drive therapeutic bone formation? To answer these questions, we will utilize two mouse models of adult bone regeneration: calvarial defect as a translational model of craniofacial bone regeneration following acute trauma and femoral marrow ablation to model rapid intramembranous bone formation in compartmentalized long bone. In Aim I we propose to characterize the Notch ligand/receptor interactions governing mesenchymal progenitor cell differentiation toward bone. In Aim II we propose to elucidate modifications and compensatory mechanisms implicated in Notch ligand deficiencies altering bone regeneration. In Aim III we propose to assess therapeutic modification of the Notch pathway to improve healing of critical-size craniofacial defects. Bone formation in wild-type and conditional Notch ligand single/double knockout mice will be evaluated following these procedures, and outcome variables will include gene expression analysis, Western blot, co-immunoprecipitation, immunohistochemistry, histomorphometry, super-resolution confocal microscopy and micro-computed tomography. Elucidation of Notch ligand/receptor interactions driving osteoblastogenesis holds significant promise for informing safe and efficacious pharmacological intervention improving bone healing. Using modern molecular biological techniques, biophysical chemistry and advanced imaging, this comprehensive cross- sectional small animal study will provide essential pre-clinical data on therapeutic ligand delivery for craniofacial bone healing. Successful completion of the proposed project will improve outcomes of reconstructive surgeries involving the skull and reduce morbidity associated with conventional bone grafting.